Analogous compounds of 6-thioguanosine triphosphate, their use in medical fields and processes for their preparation

ABSTRACT

The invention relates to analogous compounds of 6-thioguanosine triphosphate of general formula (I). A compound of the general formula (I); wherein the dashed bond in the sugar moiety can be either single or double and wherein R1, R2, R3, R4 or R5, equal or different between each other, have general formula -(Int) m -Ter, wherein m is between 0 and 12 and Int and Ter are Internal and Terminal building blocks, wherein Int is selected from the group consisting of formula (II); and Ter is selected from the group consisting of formula (III). And wherein X represents either carbon or nitrogen atom within aromatic ring, Y represents either oxygen or sulphur atom and an additional group Q, group Qi or groups Qi (Qi indicates that the group or several groups may be bound to any unsaturated moiety of the ring) are selected from the group consisting of —OH, —COOH, —N(CH 3 ) 2 , —N(CH 2 —CH 3 ) 2 |—CO—CH 3 , —CO—O—CH 3 , —O—CH 3 , —S—CH 3 , —SO 2 —CH 3 , —CN, —NO 2  or -Halogen elements, and wherein R5 may be formula (IV) and metal and ammonium salts thereof, wherein n is between O and 5, or oxygen or phosphorus is partially or completely replaced by nitrogen, sulphur, methyleno groups or their derivatives. The invention also concerns the uses of the above mentioned compounds in medical field and the process for their preparation.

The present invention relates to analogous compounds of 6-thioguanosinetriphosphate, their use in medical field and process for theirpreparation.

Particularly, the invention refers to the therapeutic use of analogouscompounds of 6-thioguanosine triphosphate for example asimmunosuppressant for the prevention of rejection of organ transplantsand of post-transplant nephropathy and in the treatment of pathologiesin which immune system is involved, such as, for instance, inflammatorychronic intestinal diseases, such as Crohn's disease, ulcerousrectocolitis, indeterminate colitis, or of auto-immune enteropathy,active chronic hepatitis, rheumatoid arthritis, Still's disease,systemic lupus erythematous, acquired haemolytic anaemia, idiopathicthrombocytopenia, polyarthritis nodosa, vasculitis, polyangitis,polymyositis, myasthenia gravis, sarcoidosis, lipoid nephritis, multiplesclerosis, dermatomyositis, pemphigus vulgaris, primary biliarycirrhosis, primary sclerosing cholangitis, recurrent multiform erythema,chronic actinic dermatitis, gangrenous hypoderm, ptyriasis rubra,Wegener's granulomatosis, cutaneous vasculitis, atopic dermatitis,psoriasis, pimply pemphigoid and, in general, in the immunosuppressivetreatment in addition to radiotherapy, corticosteroids and othercytotoxic agents. The latter also involves immunosuppressive therapyafter organ transplantation (e.g. kidney, heart, lung, pancreas andliver transplantation).

The cells involved in the inflammatory immune response are able tosurvive at the inflammatory site, however, after completion of suchresponse, the majority of cells must “die” to maintain the homeostasisof organism (Boise, 1995). Since the uncontrolled lymphocyteproliferation may cause the development of inflammatory chronicpathologies, the immune system controls the depletion of activatedlymphocytes by a process named apoptosis (programmed cell death).

This would assume a particular importance for the immune system of themucosa, since the apoptosis resistance of lamina propria cells can leadto a chronic inflammatory response at the intestinal level (Tiede,2003).

The activation of the mucosal immune system plays a key role in thepathogenesis of Crohn's disease. Particularly, pro-inflammatorycytokines produced by T lymphocytes and macrophages, in particularinterleukine-6 (IL-6) and interleukine-12 (IL-12), may cause Tlymphocytes resistance against apoptosis, which in its turn provokes anintestinal accumulation of lymphocytes and establishes a long-lastingdisease (Tiede, 2003).

The lymphocytes activation starts with two signals: the specific bindingof antigens to the TCR (T cell receptor) and a second co-stimulatorysignal represented by transmembrane proteins, such as CD28 (Maltzman,2003). It has been shown that co-stimulation with CD28 enhances in vitrosurvival of activated T lymphocytes; in fact, CD28 induces an enhancedproduction of interleukine-2 (IL-2) acting as extrinsic factor for Tlymphocytes survival, and the intrinsic ability of T lymphocytes to beresistant against apoptosis (Boise, 1995 bis). This occurs since CD28action is associated with the expression of an anti-apoptotic gene,named bcl-x_(L) gene (Khoshnan, 2000; Noel, 1996).

The steps through which an inhibition of apoptosis takes place, will besynthesized as follows, as shown in FIG. 1:

-   -   CD28 acts through its cytoplasmaic portion with a complex of        “adaptor” proteins and with a molecule, named Vav (Frauwirth,        2002);    -   Vav acts as guanosine nucleotide exchange factor (GEF) for        another molecule named Rac1 (Frauwirth, 2002);    -   Rac1, a small GTPase, in such a way switches between an inactive        state bound to GDP and an active state bound to GTP (Frauwirth,        2002);    -   Activated Rac1, in its turn, leads to the activation of kinases        (IKK) that phosphorylate the NF-κB inhibitory proteins (like        I-κB alfa) (Marinari, 2002) through MEK phosphorylation;    -   Thus, NF-κB is not anymore retained in an inactive form in the        cytosol but is able to translocate to nucleus where induces        bcl-x_(L) expression (Khoshnan, 2000);    -   Further, activated Rac1 stimulates the activation of a protein        belonging to the STAT family (Signal Transducers and Activators        of Transcription), that is STAT-3, thus inducing its        translocation to the nucleus and the corresponding expression of        STAT-3 dependent genes (Faruqi, 2001). In particular, STAT-3        induces bcl-x_(L) expression thus contributing to the resistance        against apoptosis and to the accumulation of T lymphocytes in        the inflamed mucosa during the course of Crohn's disease        (Mudter, 2003). On the other hand, the study of intestinal T        lymphocytes has pointed out that STAT-3 is steadily activated in        patients with Crohn's disease, but not in healthy voluntaries        (Lovato 2003).

Rac1, together with RhoA and Cdc42, belongs to the Rho family which is asuperfamily of small G protein characterized in that they are able tobind guanosine nucleotides and to regulate many cellular responses. Theycycle between an inactive state, when bound to GDP, to an active statewith GTP in place of GDP. This reaction is sustained upon guanosinenucleotide exchange factors named GEFs (like Vav). The binding with GTPinduces a conformational change, which allows Rac1 and other GTPases tobind to their effectors. The action of other proteins calledGTPase-activating protein (GAPs) stimulates the innate GTPase activityof these small G proteins and causes them to turn back to their GDPbound inactive state. Rac1, as in general all GTPase belonging to theRho family, plays an important role in the mitogenesis processes,proliferation, and invasivity, since it stimulates alterations of thegene expression, in the present case of the gene bcl-x_(L), modulatingthe activity of transcription factors, such as, in the present case,NF-κB and STAT-3 (Van Aelst, 1997).

Azathioprine is regarded as “gold standard” of the immunosuppressivetherapy of Crohn's disease, also if the mechanism of action of suchactive principle is still unknown. However, the inhibition of the purinenucleotide biosynthesis with suppression of DNA and RNA synthesis anddownregulation of T and B lymphocytes function (Tiede 2003), is assumedto be the main therapeutic mechanism of azathioprine.

Recently, a new mechanism of action of azathioprine acting at the Tlymphocytes level has been shown. After the evidence that azathioprineinduces in vitro apoptosis of activated T lymphocytes and that treatmentwith azathioprine causes apoptosis of both circulating and laminapropria T lymphocytes of IBD patients, the specific molecular mechanismswere pointed out (Tiede 2003).

The key point is represented by the metabolite 6-thioguanosinetriphosphate (6-thioGTP) which represents the real functional metaboliteof the drug. Specifically, 6-thioGTP binds directly to Rac1 in place ofGTP, thus blocking its activation. Such a block is highly specific forRac1, as other GTPases belonging to the same family are not inhibited by6-thioGTP, and this specificity suggests that the block would becorrelated to the structure of Rac1 protein. The fact that anaccumulation of the Rac1 guanosine nucleotide exchange factor vav wasobserved, is consistent with a compensatory mechanism to achieve Rac1activation.

The block of the activation of Rac1 would result in the block of NF-κBand STAT-3, normally induced by Rac1 itself, and thus in the block ofthe bcl-x_(L) gene expression, detected both at the mRNA and proteinlevels. Thus, azathioprine, by modulating Rac1 activity switches ananti-apoptotic co-stimulatory signal, mediated by CD28, into apro-apoptotic signal.

This new mechanism of action is able to explain the well-known “delay”in the therapeutic effect of azathioprine, which needs a long treatmenttime to elicit a clinical response in such a way that benefits andclinical response were not observed earlier than 4 months of therapy.This is due to the fact that 6-thioGPT has 20-fold less affinity to Rac1in comparison with GTP, which is normally bound to Rac1. Therefore, theazathioprine treatment needs the simultaneous and prolongedadministration with high steroids doses, having remarkable side effectssuch as osteoporosis, diabetes, cataract.

In the light of the above it would be desirable to have at disposal newimmunosuppressive drugs eliciting a faster and more efficacioustherapeutic response in comparison with the already known compounds.

According to the present invention, a new class of 6-thioGTP analogousdrugs able to inhibit Rac1 and characterized by greater affinity toRac1, an higher suppression of Rac1 activity and, thus having a greaterimmunosuppressive power and action in comparison with the delayed effectof azathioprine treatment and a best therapeutic effect, was prepared.

It is therefore an object of the present invention to provide a class ofanalogous compounds of 6-thioguanosine triphosphate of general formula(I):

wherein the dashed bond in the sugar moiety can be either single ordouble and wherein R₁, R₂, R₃, R₄ or R₅, equal or different between eachother, have general formula -(Int)_(m)-Ter, wherein m is between 0 and12 and Int and Ter are Internal and Terminal building blocks, whereinInt is selected from the group consisting of

and Ter is selected from the group consisting of

-   -   wherein X represents either carbon or nitrogen atom within        aromatic ring, Y represents either oxygen or sulphur atom and an        additional group Q, group Qi or groups Qi (Qi indicates that the        group or several groups may be bound to any unsaturated moiety        of the ring) are selected from the group consisting of —OH,        —COOH, —N(CH₃)₂, —N(CH₂—CH₃)₂, —CO—CH₃, —CO—O—CH₃, —O—CH₃,        —S—CH₃, —SO₂—CH₃, —CN, —NO₂ or -Halogen elements.    -   Alternatively R₅ may be

-   -   and metal and ammonium salts thereof, wherein n is between 0 and        5, or oxygen or phosphorus is partially or completely replaced        by nitrogen, sulphur, methylene groups or their derivatives.    -   In some embodiments (particularly, but not limited to those        where R₅ is HO—[PO₃]_(n), and metal and ammonium salts thereof,        wherein n is between 0 and 5, or oxygen or phosphorus is        partially or completely replaced by nitrogen, sulphur, methylene        groups or their derivatives) TER may be selected from the group        consisting of:

Compounds of formula (I) can be labelled, particularly with R₃ or R₄selected from

wherein Q is selected from —OH (FAM) or —N(CH₃)₂ (TAMRA).

In addition the sugar moiety of compounds of formula (I) can be selectedfrom the group consisting of the following sugar moieties or sugar-likemoieties:

Compounds of the invention may be of the general formula (II) derivedfrom general formula (Ia) and/or (I) where R₁ is [—SH] (it is understoodthat the Guanosine moiety may undergo keto-enol tautomeric shifts and sogive rise to [═S]), R₂ is [—H], R₅ is [—(PO₃)_(n)—OH] and one of R₃ andR₄ is [—OH] and the other of R₃ and R₄ is [—O—CO—NH-Int_(m)-Ter] (bothversions are provided in the same mixture, i.e., [—O—CO—NH-Int_(m)-Ter]:will be the same, but a proportion of the molecules will have[—O—CO—NH—-Int_(m)-Ter]: attached at R₃ with R₄ being OH, and anotherproportion of the molecules will have [—O—CO—NH-Int_(m)-Ter]: attachedat R₄ with R₃ being OH):

wherein n=1, 2 or 3, m is between 0 and 5, Int is selected from thegroup consisting of

and Ter is selected from the consisting of

wherein X represents either carbon or nitrogen atom within aromaticring, Y represents either oxygen or sulphur atom and an additional groupQ or groups Qi (i indicating the position of any unsaturated moiety ofthe ring to which the group Q may be bound) are selected from the groupconsisting of —CH₃, —C(CH₃)₃, —OH, —COOH, —CO—CH₃, —CO—O—CH₃, —O—CH₃,—S—CH₃, —SO₂—CH₃, —N(CH₃)₂, —N(CH₂—CH₃)₂, —CN, —NO₂ or -Halogenelements.

In some embodiments of the invention of formula (I), (Ia) and (II), Teris selected from the group consisting of

According to some embodiments of the present invention the compounds offormula (I) are the compounds described below:

wherein R₁, R₂, R₃, R₄ or R₅, equal or different between each other,have general formula -(Int)_(m)-Ter, wherein m is between 0 and 12 andInt and Ter are Internal and Terminal building blocks, wherein Int isselected from the consisting of

and Ter is selected from the consisting of

wherein an additional group Q, group Qi or groups Qi (Qi indicates thatthe group or several groups may be bound to any unsaturated moiety ofthe ring) are selected from the group consisting of —OH, —COOH,—N(CH₃)₂, —N(CH₂—CH₃)₂ or -Halogen elements

In addition the sugar moiety of compounds of formula (I) can be selectedfrom the group consisting of the following sugar moieties or sugar-likemoieties:

Compounds of formula (I), (Ia) and (II) can be labelled, particularlywith R₃ or R₄ selected from

wherein Q is selected from —OH (FAM) or —N(CH₃)₂ (TAMRA).

According to some embodiments of the present invention the compounds offormula (I), (Ia) and (II) are the compounds described below:

-   -   SM4410

2′,3′-EDA-6-Thio-GTP, ID: 05B-0

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 6 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R3 o —R4 —O—

—NH— —CH₂— —CH₂— —NH— —H m = 0 Ter —R3 o —OH —R4 m = 0 Ter —R2 —H

FAM-2′,3′-EDA-6-Thio-GTP

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 0 Ter—R2 —H m = 8 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R3 o —R4 —O—

—NH— —CH₂— —CH₂— —NH—

m = 0 Ter —R3 o —OH R4

TAMRA-2′,3′-EDA-6-Thio-GTP

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 0 Ter—R3 o —OH R4 m = 8 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ —R3 o —R4 —O—

—NH— —CH₂— —CH₂— —NH—

m = 8 Int₈ Ter —R3 o —R4

m = 0 Ter —R2 —H

Aspartate-2,3′-EDA-6-Thio-GTP, ID: 05B-1

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 9 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Ter —R3 o —R4 —O—

—NH— —CH₂— —CH₂— —NH—

—CH₂— —COOH m = 0 Ter —R2 m = 0 Ter —R3 o —R4

Glutamate-2′,3′-EDA-6-Thio-GTP, ID: 05B-2

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 10 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Int₁₀ Ter —R3 o —R4 —O—

—NH— —CH₂— —CH₂— —NH—

—CH₂— —CH₂— —COOH m = 0 Ter —R2 —H m = 0 Ter —R3 o —OH —R4

Threonine-2′,3′-EDA-6-Thio-GTP, ID: 05B-3

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 9 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Ter —R3 o —R4 —O—

—NH— —CH₂— —CH₂— —NH—

—CH₃ m = 0 Ter —R2 —H m = 0 Ter —R3 o —OH —R4

Serine-2′,3′-EDA-6-Thio-GTP

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 9 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Ter —R3 o —R4 —O—

—NH— —CH₂— —CH₂— —NH—

—CH₂— —OH m = 0 Ter —R3 o —OH —R4 m = 0 Ter —R2 —H

2′,3′,5′,O-Triacetyl-N-2-(Acetyl-6″-aminohexyl)-guanosine

m = 0 Ter —R1 —OH m = 2 Int₁ Int₂ Ter —R5 —O—

—CH₃ m = 2 Int₁ Int₂ Ter —R3 e —R4 —O—

—CH₃ m = 8 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —CH₂— —NH—

—CH₃

2′,3′,5′-Triacetyl-N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine (V4)[TWI 107/7]

m = 0 Ter —R1 —SH m = 2 Int₁ Int₂ Ter —R5 —O—

—CH₃ m = 2 Int₁ Int₂ Ter —R3 e —R4 —O—

—CH₃ m = 8 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —CH₂— —NH—

—CH₃

N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine

m = 0 Ter —R1 —SH m = 0 Ter —R5 —OH m = 0 Ter —R3 e R4 —OH m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —CH₂——CH₂— —NH—

—CH₃

N-2-(6″-Aminohexyl)-6-Thioguanosine, ID: 05A-0

m = 0 Ter —R1 —SH m = 0 Ter —R3 e —OH —R4 m = 0 Ter —R5 —OH m = 6 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —CH₂— —CH₂——NH₂

N-2-(6″-guanidino-hexyl)-6-Thioguanosine, ID: 05A-1

m = 0 Ter —R1 —SH n = 0 Ter —R5 —OH m = 0 Ter —R3 e —OH —R4 m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —CH₂——CH₂— —NH—

—NH₂

N-2-(6″-Aminohexyl)-6-Thio-GMP

m = 0 Ter —R1 —SH n = 1 1 Ter —R5 —PO₃— —OH m = 0 Ter —R3 e —OH —R4 m =6 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —CH₂——CH₂— —NH₂

N-2-(6″-guanidino-hexyl)-6-Thio-GMP, ID: 05A-2

m = 0 Ter —R1 —SH n = 1 1 Ter —R5 —PO₃— —OH m = 0 Ter —R3 e —OH —R4 m =8 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —NH—

—NH₂

N-2-(6″-Aminohexyl)-6-Thio-GTP

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 0 Ter—R3 e —OH —R4 m = 6 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R2 —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —CH₂— —NH₂

N-2-(6″-guanidino-hexyl)-6-Thio-GTP, ID: 05A-3

m = 0 Ter —R1 —SH n = 3 1 2 3 Ter —R5 —PO₃— —PO₃— —PO₃— —OH m = 0 Ter—R3 e —OH —R4 m = 8 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2—CH₂— —CH₂— —CH₂— —CH₂— —CH₂— —CH₂— —NH—

—NH₂

N-2-(6″-Aspartate-hexyl)-6-Thioguanosine

m = 0 Ter —R1 —SH n = 0 Ter —R5 —OH m = 0 Ter —R3 e —OH —R4 m = 10 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Int₁₀ Ter —R2 —CH₂— —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —NH—

—CH₂— —COOH

N-2-(6″-Glutamate-hexyl)-6-Thioguanosine

m = 0 Ter —R1 —SH n = 0 Ter —R5 —OH m = 0 Ter —R3 —OH e —R4 m = 11 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Int₁₀ Int₁₁ Ter —R2 —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —CH₂— —NH—

—CH₂— —CH₂— —COOH

N-2-(6″-Threonine-hexyl)-6-Thioguanosine

m = 0 Ter —R1 —SH n = 0 Ter —R5 —OH m = 0 Ter —R3 o —OH —R4 m = 10 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Int₁₀ Ter —R2 —CH₂— —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —NH—

—CH₃

N-2-(6″-Serine-hexyl)-6-Thioguanosine

m = 0 Ter —R1 —SH n = 0 Ter —R5 —OH m = 0 Ter —R3 e —OH —R4 m = 10 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Int₁₀ Ter —R2 —CH₂— —CH₂— —CH₂——CH₂— —CH₂— —CH₂— —NH—

—CH₂— —OH

N-2-(6″-Aminobutyl)-6-Thio-Guanosine, ID: 05C-0

m = 0 Ter —R1 —SH n = 0 Ter —R5 —OH m = 0 Ter —R3 e —OH —R4 m = 4 Int₁Int₂ Int₃ Int₄ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —NH₂

2-N-2-(6″-guanidino-butyl)-6-Thioguanosine, ID: 05C-1

m = 0 Ter —R1 —SH n = 0 Ter —R5 —OH m = 0 Ter —R3 e —OH —R4 m = 6 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —NH—

—NH₂

2-N-2-(6″-guanidino-butyl)-6-Thio-GMP, ID: 05C-2

m = 0 Ter n = 1 1 Ter m = 0 Ter —R1 —SH —R5 —PO₃— —OH —R3 e —R4 —OH m =6 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —NH—

—NH₂

2-N-2-(6″-guanidino-butyl)-6-Thio-GTP, ID: 05C-3

m = 0 Ter n = 3 1 2 3 Ter m = 0 Ter —R1 —SH —R5 —PO₃— —PO₃— —PO₃— —OH—R3 e —R4 —OH m = 6 Int₁ Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R2 —CH₂— —CH₂——CH₂— —CH₂— —NH—

—NH₂

N-2-(6″-Aspartate-butyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —NH—

—CH₂— —COOH

N-2-(6″-Glutamate-butyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 9 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂——NH—

—CH₂— —CH₂— —COOH

N-2-(6″-Threonine-butyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —NH—

—CH₃

N-2-(6″-Serine-butyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂— —CH₂— —CH₂— —NH—

—CH₂— —OH

N-2-(6″-Aminopropyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 0 Int₁Int₂ Int₃ Ter —R2 —CH₂— —CH₂— —CH₂— —NH₂

N-2-(6″-guanidino-propyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 5 Int₁Int₂ Int₃ Int₄ Int₅ Ter —R2 —CH₂— —CH₂— —CH₂— —NH—

—NH₂

N-2-(6″-Aspartate-propyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂— —CH₂— —CH₂— —NH—

—CH₂— —COOH

N-2-(6″-Glutamate-propyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —CH₂— —CH₂— —NH—

—CH₂— —CH₂— —COOH

N-2-(6″-Threonine-propyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂— —CH₂— —CH₂— —NH—

—CH₃

N-2-(6″-Serine-propyl)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂— —CH₂— —CH₂— —NH—

—CH₂— —OH

N-2-(6″-Amino-2-butene)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 3 Int₁Int₂ Int₃ Ter —R2 —CH₂—

—CH₂— —NH₂

N-2-(6″-guanidino-2-butene)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 5 Int₁Int₂ Int₃ Int₄ Int₅ Ter —R2 —CH₂—

—CH₂— —NH—

—NH₂

N-2-(6″-Aspartate-2-butene)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —R4 —OH m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂—

—CH₂— —NH—

—CH₂— —COOH

N-2-(6″-Glutamate-2-butene)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂—

—CH₂— —NH—

—CH₂— —CH₂— —COOH

N-2-(6″-Threonine-2-butene)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂—

—CH₂— —NH—

—CH₃

N-2-(6″-Serine-2-butene)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂—

—CH₂— —NH—

—CH₂— —OH

N-2-(6″-Amino-2-butyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 3 Int₁Int₂ Int₃ Ter —R2 —CH₂— —C≡C— —CH₂— —NH₂

N-2-(6″-guanidino-2-butyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 5 Int₁Int₂ Int₃ Int₄ Int₅ Ter —R2 —CH₂— —C≡C— —CH₂— —NH—

—NH₂

N-2-(6″-Aspartate-2-butyne)-6-Thioguanosine

M = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂— —C≡C— —CH₂— —NH—

—CH₂— —COOH

N-2-(6″-Glutamate-2-butyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —C≡C— —CH₂— —NH—

—CH₂— —CH₂— —COOH

N-2-(6″-Threonine-2-butyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂— —C≡C— —CH₂— —NH—

—CH₃

N-2-(6″-Serine-2-butyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 7 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Ter —R2 —CH₂— —C≡C— —CH₂— —NH—

—CH₂— —OH

N-2-(6″-Amino-2,4-hexadiyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 4 Int₁Int₂ Int₃ Int₄ Ter —R2 —CH₂— —C≡C— —C≡C— —CH₂— —NH₂

N-2-(6″-guanidino-2,4-hexadiyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 6 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Ter —R2 —CH₂— —C≡C— —C≡C— —CH₂— —NH—

—NH₂

N-2-(6″-Aspartate-2,4-hexadiyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —C≡C— —C≡C— —CH₂— —NH—

—CH₂— —COOH

N-2-(6″-Glutamate-2,4-hexadiyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 9 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Int₉ Ter —R2 —CH₂— —C≡C— —C≡C— —CH₂——NH—

—CH₂— —CH₂— —COOH

N-2-(6″-Threonine-2,4-hexadiyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —C≡C— —C≡C— —CH₂— —NH—

—CH₃

N-2-(6″-Serine-2,4-hexadiyne)-6-Thioguanosine

m = 0 Ter n = 0 Ter m = 0 Ter —R1 —SH —R5 —OH —R3 e —OH —R4 m = 8 Int₁Int₂ Int₃ Int₄ Int₅ Int₆ Int₇ Int₈ Ter —R2 —CH₂— —C≡C— —C≡C— —CH₂— —NH—

—CH₂— —OH

Further compounds of the invention are disclosed, with reference titlesindicated.

Compounds according to the present invention, can be advantageously usedin medical field; therefore another object of the present invention arepharmaceutical composition comprising at least one of the compounds ofthe above mentioned formula (I) as active principle and one or morepharmaceutically acceptable co-adjuvants or excipients, that are knownto those skilled in the art and currently in use in the pharmaceuticaltechnology.

It is an object of the invention to provide for compounds for thepreparation of an immunosuppressive drug. Its uses and therapeutical andmedical uses thereof. for It is an object of the present invention toprovide the compounds and compositions of the invention, and their uses,in the following methods of treatment, and/or therapy; the prevention ofrejection of organ transplants (e.g. kidney, heart, lung, pancreas,liver transplantation) and of post-transplant nephropathy and in thetreatment of pathologies in which immune system is involved, such as,for instance, inflammatory chronic intestinal diseases, such as Crohn'sdisease, ulcerous rectocolitis, indeterminate colitis, or auto-immuneenteropathy, active chronic hepatitis, rheumatoid arthritis, Still'sdisease, systemic lupus erythematous, acquired haemolytic anaemia,idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis,polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoidnephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris,primary biliary cirrhosis, primary sclerosing cholangitis, recurrentmultiform erythema, chronic actinic dermatitis, gangrenous hypoderm,ptyriasis rubra, Wegener's granulomatosis, cutaneous vasculitis, atopicdermatitis, psoriasis, pimply pemphigoid and, in general, in theimmunosuppressive treatment in addition to radiotherapy, corticosteroidsand other cytotoxic agents. In addition, the compounds according to thepresent invention can be advantageously used for the preparation of amedicament for the treatment of cancer.

The present invention further relates to the use of labelled compoundsof formula (I), particularly with R₃ or R₄ selected from

wherein Q is selected from —OH (FAM) or —N(CH₃)₂ (TAMRA) as probes forthe evaluation of the binding properties of the compounds of formula (I)by the RacI/Vav system.

According to a further aspect, the present invention refers to a processfor the preparation of compounds of formula (I), (Ia) and (II), whereinthe introduction of the —NH—R group at the 2 position of guanosine ringcomprises the following steps:

a) protection of the NH moiety of tri-O-acetyl-inosine;b) oxidative guanosine ring-opening and O-deprotection;c) guanosine ring-closing and introduction of a SH group at the 2position of the guanosine ring through the use of CS₂;d) replacing the SH group at the 2 position with an amino-linker byusing an excess of an aliphatic diamine. The process may furthercomprise an additional step e) of protection of ribose OH groups and ofthe primary amine group by acetylation, and, moreover, an additionalstep f) of thiolation of C═O groups through the use of Lawesson'sreagent.

The present invention will be now described, for illustrative but notlimitative purposes, according to its preferred embodiments, withparticular reference to the Figures of the enclosed drawings.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the scheme of the mechanism of action of the apoptosisinhibition and of the action of azathioprine.

FIG. 2 shows the apoptosis induction in human CD4+ T Lymphocytes byV1=BMB20=EDA-6-Thio-GTP, V2=TWI 35/1=N-2-(6″-Aminohexyl)-guanosine,V3=TWI 71/2=2′,3′,5′,o-Triacetyl-N-2-(Acetyl-6″-aminohexyl)-guanosine,V4=TWI107/7=2′,3′,5′-Triacetyl-N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine,V5=BMB=TAMRA-EDA-6-Thio-GTP after 3 days.

FIG. 3 shows the apoptosis induction in human CD4+ T Lymphocytes byV1=BMB20=EDA-6-Thio-GTP, V2=TWI 35/1=N-2-(6″-Aminohexyl)-guanosine,V3=TWI 71/2=2′,3′,5′,o-Triacetyl-N-2-(Acetyl-6″-aminohexyl)-guanosine,V4=TWI107/7=2′,3′,5′-Triacetyl-N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine,V5=BMB=TAMRA-EDA-6-Thio-GTP after 4 days.

FIG. 4 shows the apoptosis induction in human CD4+ T Lymphocytes byV1=BMB20=EDA-6-Thio-GTP, V2=TWI 35/1=N-2-(6″-Aminohexyl)-guanosine,V3=TWI 71/2=2′,3′,5′,o-Triacetyl-N-2-(Acetyl-6″-aminohexyl)-guanosine,V4=TWI107/7=2′,3′,5′-Triacetyl-N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine,V5=BMB=TAMRA-EDA-6-Thio-GTP after 5 days.

FIG. 5: Induction of apoptosis. Buffy stands for “buffy coat” which isthe fraction of blood obtainable by centrifugation and containingleukocytes and platelets. In this case buffy coats were used to isolatemonocytes for the experiments. “Buffy 1 vom 08/03/05” means “Buffy coat1 of Mar. 8, 2005”. V1 and V3 have been tested 4 times in fourindependent experiments, while V2 and V5 were tested twice in twoseparate experiments. For instance: V1 was tested twice on Aug. 3, 2005(where Aug. 3, 2004 is written this is in error and should read08/03/2005) and twice on 22/04/2005

FIG. 6: Results of biological activity of compounds compiled in FIG. 5.

FIG. 7: list of further compounds of the invention, as well as providingkey to some of the shorthand used in identifying some of the molecules.It will be noted that some compounds have different enantiomeric forms,and the representations in FIG. 7 may show an alternative enantiomericform than that discussed in the text.

FIG. 8: Caspase-graph: Luminescence value of untreated cells was definedas 100% Caspase activity. Due to their specific luminescence value thecaspase activity of treated cells were calculated accordingly. Inductionof Caspase activity_(x)[%]=Caspase activity_(x)[%]−Caspaseactivity_(untreated)[%]. Means±SEM (Standart Error of the Mean=Standarddeviation/radical(n)) of three different experiments are presented.Summary of this graph: 6-thio-GTP was able to induce caspase activity(as positive control). 05B-0 was also very effective. 05B-1 was lesseffective than 05B-0 in agreement with the previous data onAnnexinV/PI-staining. Concerning Group-D derivatives 06D-13, 06D-14 andto a lesser extent 06D-22 were promising.

EXAMPLE 1 Process for the Preparation of 2-substituted-6-thio-guanosineNucleotides Preparation of2′,3′,5′-Tri-O-acetyl-1-[(2-methoxyethoxy)methyl] inosine

(Kohyoma et al., 2003)

Inosine-2′,3′,5′-triacetate (4 g, 10.2 mmol) was dissolved in 100 mldichloromethane and the solution was treated with 1.4 ml (12.2 mmol)2-methoxyethoxymethylchloride at 0° C. in the presence ofdiisopropylethylamine as a supportive base. After 1 hour the reactionwas quenched with water. The solution was stirred for 30 min andchloroform was added afterwards.

The aqueous layer was extracted with chloroform and the combined andwashed organic layers were concentrated to dryness. The reaction yielded3.93 g (8.2 mmol, 80%) of2′,3′,5′-Tri-O-acetyl-1-[(2-methoxyethoxy)methyl] inosine afterchromatographic purification (silica gel, EtOAc-MeOH, 50:1).

Preparation of 5-Amino-1-O-ribofuranosylimidazole-4-carboxamide (Kohyomaet al., 2003)

Aqueous ammonia solution (28%, 20 ml) was added to a solution of2′,3′,5′-Tri-O-acetyl-1-[(2-methoxyethoxy)methyl] inosine (3.5 g, 7.3mmol) in 50 ml methanol. The reaction mixture was stirred for 1 hour atroom temperature and concentrated, yielding the deprotected nucleoside(2.44 g, 6.9 mmol, 95%). This product was used in the next step withoutfurther purification.

The nucleoside (2.44 g, 6.9 mmol) was refluxed with 50 ml aqueous sodiumhydroxide (0.2 M) for 1 hour, cooled to room temperature, neutralizedwith HCl (6 M) and evaporated to dryness. The residue was dissolved inethanol, filtered from insoluble material and concentrated to dryness.Purification of the crude product by column chromatography (silica gel,CHCl₃:MeOH, 3:1) gave 1.32 g (5.11 mmol, 70%) of5-Amino-1-β-ribofuranosylimidazole-4-carboxamide.

Preparation of 2-Mercaptoinosine (Imai et al., 1971)

The 5-Amino-1-β-ribofuranosylimidazole-4-carboxamide (1.3 g, 5 mmol) wasdissolved in pyridine and 18 ml (15 mmol) phenyl-isothiocyanate wereadded slowly. The reaction mixture was refluxed for 2 hours under argonatmosphere. The solution was cooled to room temperature, the precipitatecollected by filtration and washed with diethyl ether. The obtainedpyridinium salt of the product was dissolved in aqueous sodium hydroxide(15%, 40 ml). The solution was heated 30 minutes at 60° C. andconcentrated under vacuum. Methanol was then added and the solution waskept in the refrigerator overnight. After 20 hours the precipitatedcolourless prisms were collected by filtration to give 1.2 g (80%)2-Mercaptoinosine.

According to an alternative method of preparation,compound-Amino-1-β-ribofuranosylimidazole-4-carboxamide (1.3 g, 5 mmol)was added to a solution of sodium hydroxide (1 g, 25 mmol) in 20 ml ofmethanol at 30° C. Carbon disulfide (1.9 g, 25 mmol) was added, and thesolution was heated in an autoclave at 180° C. for 3 hours. The mixturewas cooled to room temperature, the precipitate was filtered off, washedwith cold methanol and recrystallized from water to yield 1.1 g (3.8mmol, 75%) 2-Mercaptoinosine.

Preparation of N-2-(6″-aminohexyl)-Guanosine

2-Mercaptoinosine (1 g, 3 mmol) was dissolved in 100 ml water and cooledto 0° C. A clear solution was obtained after ultrasonic irradiation.Hydrogen peroxide (1 ml, 9 mmol) was added within 20 min under vigorousstirring. After 1 hour of stirring at 0° C., HPLC chromatographyindicated that the starting compound has been completely oxidized to thesodium salt of Inosine-2-sulfonic acid. Without further purification,the resulting solution was treated with an excess of 1,6-diaminohexane(20 g, 200 mmol). The mixture was refluxed for 2.5 hours at 155° C. Theexcess of 1,6-diaminohexane was removed by vacuum distillation to givean orange residue. N-2-(6″-Aminohexyl)-guanosine (0.85 g, 2.2 mmol, 50%)was obtained after purification of the crude product by columnchromatography (silica gel RP-18, linear gradient from 100% water to100% methanol). Preparation of2′,3′,5′-triacetyl-N-2-(6″-acetamide-hexyl)-guanosine (Ostermann et al.,1999)

A solution of nucleoside (220 mg, 0.575 mmol) in 20 ml dry pyridine wasstirred with 1 ml (10 mmol) acetic anhydride at room temperature for 15hours under argon atmosphere. The solvent was removed under vacuum andthe residue dissolved in a mixture of CHCl₃ (10 ml) and CH₃OH (2 ml).This solution was loaded on silica gel and eluted with CHCl₃/CH₃OH (5:1)to give 284.7 mg (0.52 mmol, 90%) of fully protected nucleoside.

Preparation of2′,3′,5′-triacetyl-N-2-(6″-thioacetamido-hexyl)-6-thioguanosine

Dioxane (200 ml) was added to the fully protected nucleoside (5.03 g,9.13 mmol). After addition of 8 g (19.8 mmol) of Lawesson's reagent thesuspension was vigorously stirred for 2 hours at 80° C. The initiallyopaque reaction mixture became clear after 10 minutes. The solution wascooled at room temperature and the solvent was evaporated by vacuumdistillation. Purification of the raw product by column chromatography(silica gel, CHCl₃), resulted in2′,3′,5′-triacetyl-N-2-(6″-thioacetamido-hexyl)-6-thioguanosine (2.92 g,5.02 mmol) in 55% yield.

EXAMPLE 2 Process for the Preparation of Analogous of ribose-modified6-thio-guanosine-triphosphate Preparation of6-Thio-Guanosine-Triphosphate (Ludwig, 1981)

Under argon atmosphere, 6-thio-guanosine (1 g, 3.34 mmol) was dissolvedin 6 ml trimethylphosphate. The solution was cooled to 0° C. and 1.3 mlof Lutidine were added. After 10 minutes, 0.4 ml (4.4 mmol) phosphorousoxychloride was carefully added to the solution. After 1 hour the excessof POCl₃ was removed under vacuum within ten minutes.

The solution of the initially formed intermediate dichlorophosphate wasthen treated with a solution of tri-n-butyl ammonium pyrophosphate (17ml, 100 mM) in dimethylformamide. After 2 minutes, the reaction wasquenched by adding 100 ml of 0.25 M triethylammonium bicarbonate buffer.Purification by ion exchange chromatography gave6-Thio-Guanosine-Triphosphate (0.5 g, 1 mmol, 30%).

Preparation of 2′/3′-EDA-6-Thio-Guanosine-triphosphate

The dry tributylammonium salt of compound (0.5 mmol) was treated with500 mg carbonyldiimidazole in 25 ml dimethylformamide. The resultingmixture was stirred for 6 hours at 0° C., brought to room temperatureand 0.2 ml methanol and subsequently 0.3 ml ethylene diamine were added.

The resulting precipitate was centrifuged down and dissolved in water.The solution was adjusted to pH 2, in order to decompose the resultingintermediate phosphoramidate at the triphosphate moiety. After 18 hours,pH was adjusted to 7.5 and the solvent was subsequently removed underreduced pressure. Purification of the crude product by ion exchangechromatography gave 116 mg (0.4 mmol, 80%)2′/3′-EDA-6-Thio-Guanosine-triphosphate.

Preparation of TAMRA-2′/3′-EDA-6-Thio-Guanosine-Triphosphate

The N-hydroxy-succinimide ester of TAMRA (1 mg, 2 μmol) was dissolved in200 μl of dry dimethylformamide and added to a solution of 1 mg (3 μmol)of 2′/3′-EDA-6-Thio-Guanosine-Triphosphate in 500 μl of 100 mM sodiumborate buffer (pH 8.5) at room temperature. After 2 hours, the reactionmixture was quenched with methanol. The TAMRA labelled product wasobtained in 70% yield (2.4 mg, 1.4 μmol) after workup of the reactionmixture by reversed phase HPLC.

EXAMPLE 3 Synthesis of 2-Substituted 6-Thio-Guanosine nucleotidesPreparation of N-2-(6″-Thioacetamido-hexyl)-6-Thioguanosine (10)

A, 7 M solution of ammonia in methanol (70 ml) was added to the fullyprotected nucleoside 9 (1.15 g, 1.97 mmol). The solution was stirred atroom temperature for 20 hours. The solvent was removed by distillationto give an off white residue. The crudeN-2-(6″-thioacetamido-hexyl)-6-Thioguanosine (10) (0.85 g, 1.8 mmol,95%) was used without further purification.

Preparation of N-2-(6″-Aminohexyl)-6-Thioguanosine (11)

A solution of nucleoside 10 (0.1 g, 0.22 mmol) in 5 ml NH₃/H₂O (30%) wasstirred for 90 minutes at 80° C. The solvent was removed in vacuo andthe residue was dissolved in water (2 ml).N-2-(6″-Aminohexyl)-6-Thioguanosine (11) (0.80 g, 2.0 mmol, 91%) wasobtained after purification of the crude product by columnchromatography (silica gel RP-18, linear gradient from 100% water to100% methanol).

Preparation of N-2-(6″-Guanidino-hexyl)-6-Thioguanosine (12)

N-2-(6″-Aminohexyl)-6-Thioguanosine (11) (0.091 g, 0.23 mmol) wasdissolved in 1 ml DMF and the resulting solution was treated with 0.34 g(0.23 mmol) Triazole-1-carboxamidine hydrochloride at room temperaturein the presence of 39 μl (0.23 mmol) N-Ethyl-diisopropylamine acting, asa supportive base. After 2 hours, the reaction mixture was evaporated todryness. Purification of the crude product by reversed phasechromatography (silica gel RP 18, linear gradient from 100% H₂O to 100%ACN) gave 0.92 g (0.21 mmol, 90%) ofN-2-(6″-Guanidino-hexyl)-6-Thioguanosine (12).

Preparation of N-2-(6″-Aminohexyl)-6-Thioguanosine-5′-monophosphate (13)

Under argon atmosphere, nucleoside 9 (0.3 g, 0.6 mmol) was dissolved in3 ml trimethylphosphate. The solution was cooled to 0° C. and treatedwith 0.3 ml of Lutidine.

After 10 minutes, 0.15 ml (1.1 mmol) phosphorous oxychloride wascarefully added. After 1 hour, the excess of POCl₃ was removed in vacuowithin ten minutes.

The solution was quenched by adding 100 ml of 0.25 M (pH 7.5)triethylammonium bicarbonate buffer. Purification by ion exchangechromatography gaveN-2-(6″-thioacetamido-hexyl)-6-Thioguanosine-5′-monophosphate. Theproduct was dissolved in 5 ml NH₃/H₂O (30%) and stirred for 90 minutesat 80° C. The solvent was subsequently removed in vacuo.N-2-(6″-Aminohexyl)-6-Thioguanosine-5′-monophosphate (13) (0.17 g, 0.36mmol, 60%) was obtained after purification of the crude product by ionexchange chromatography.

Preparation of N-2-(6″-Guanidino-hexyl)-6-Thioguanosine-5′-monophosphate(14)

N-2-(6″-Aminohexyl)-6-Thioguanosine-5′-monophosphate (13) (0.053 g, 0.11mmol) was dissolved in a mixture of 0.8 ml water and 0.5 ml DMF. Thesolution was subsequently treated with 0.16 g (0.11 mmol)Triazole-1-carboxamidine hydrochloride at room temperature in thepresence of 18 μl (0.11 mmol) N-Ethyl-diisopropylamine, acting as asupportive base. After 16 hours, the reaction mixture was concentratedto dryness. Purification of the crude product by reversed phasechromatography (silica gel RP 18, linear gradient from 100% H₂O to 100%ACN) gave 0.037 g (0.072 mmol, 65%) ofN-2-(6″-Guanidino-hexyl)-6-Thioguanosine-5′-monophosphate (14).

EXAMPLE 4 Synthesis of Ribose-Modified 6-Thio-GTP Analogs Preparation ofAspartate-2′/3′-EDA-6-Thio-Guanosine-Triphosphate (4)

The N-hydroxy-succinimide ester of Boc/tBu protected Aspartate (2.8 mg,7 μmol) was dissolved in 200 μl of dry DMF and added to a solution of 1(5 mg, 7 μmol) in 500 μl of 100 mM sodium borate buffer (pH 8.5) at roomtemperature. After 16 hours, the reaction mixture was quenched withmethanol. The Aspartate derivative 4 was obtained in 70% yield (3.6 mg,4.9 μmol) after workup of the reaction mixture by reversed phase HPLC.

Preparation of Glutamate-2′/3′-EDA-6-Thio-Guanosine-Triphosphate (5)

The N-hydroxy-succinimide ester of Boc/tBu-protected Glutamate (2.9 mg,7 μmol) was dissolved in 200 μl of dry DMF and added to a solution of 1(5 mg, 7 μmol) in 500 μl of 100 mM sodium borate buffer (pH 8.5) at roomtemperature. After 16 hours, the reaction mixture was quenched withmethanol. The Glutamate labeled product 5 was obtained in 70% yield (3.7mg, 4.9 μmol) after workup of the reaction mixture by reversed phaseHPLC.

Preparation of Threonine-2′/3′-EDA-6-Thio-Guanosine-Triphosphate (6)

The N-hydroxy-succinimide ester of Boc-protected Threonine (2.3 mg, 7μmol) was dissolved in 200 μl of dry DMF and added to a solution of 1 (5mg, 7 μmol) in 500 μl of 100 mM sodium borate buffer (pH 8.5) at roomtemperature. After 16 hours, the reaction mixture was quenched withmethanol. The Threonine derivative 6 was obtained in 70% yield (3.4 mg,4.9 μmol) after workup of the reaction mixture by reversed phase HPLC.

EXAMPLE 5 Analysis of the Ability of 5 New Synthesized6-Thio-GTP-Derivatives to Induce Apoptosis in Human CD4⁺ T LymphocytesUsed Substances:

-   -   Azathioprine    -   6-Mercaptopurine    -   V1=BMB20=EDA-6-Thio-GTP    -   V2=TWI 35/1=N-2-(6″-Aminohexyl)-guanosine    -   V3=TWI        71/2=2′,3′,5′,o-Triacetyl-N-2-(Acetyl-6″-aminohexyl)-guanosine    -   V4=TWI        107/7=2′,3′,5′-Triacetyl-N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine    -   V5=BMB=TAMRA-EDA-6-Thio-GTP

V3 and V4 were not soluble in water, therefore V3 was reconstituted withethanol and V4 was reconstituted with methanol.

Protocol:

Human peripheral blood mononuclear cells (PBMC) from 4 buffycoats wereisolated using Ficoll-Hypaque gradients. PBMC were further purifiedusing CD4 monoclonal antibodies attached to immunomagnetic microbeadsaccording to the protocol provided by the manufacturer (MiltenyiBiotec). T lymphocytes were stimulated in complete RPMI-1640 medium(RPMI-1640+10% FCS+100 U/ml Penicillin/Streptomycin+3 mM L-Glutamin) for3, 4 or 5 days with coated antibodies to CD3 (0.04 μg/ml) and solubleCD28 antibodies (PharMingen; 1 □g/ml) plus IL-2 (R & D Systems,Wiesbaden, Germany; 40 U/ml). Azathioprine, 6-Mercaptopurine, V1, V2,V3, V4 or V5 were added to the T cell cultures at day 0 at a finalconcentration of 5 μM. To determine induction of apoptosis in these Tlymphocytes, cells were analyzed by FACS. For FACS analysis, apoptoticcells were detected by staining with annexin V and propidium iodideusing the Annexin V FITC Apoptosis Detection Kit I (PharMingen). Inbrief, T cells were washed twice in PBS, and the pellet was resuspendedin annexin V binding buffer (PharMingen) at a concentration of 10⁶ cellsper milliliter. Annexin V FITC and propidium iodide were added (5 μl ofeach per 10⁵ cells), Samples were gently mixed and incubated for 15minutes at room temperature in the dark before FACS analysis.

Results:

-   -   Annexin-positive, propidium iodide-negative cells (black bars)        present the rate of early apoptotic cells. Annexin-positive,        propidium iodide-positive cells (white bars) present late        apoptotic or necrotic cells.    -   Induction of apoptosis=(Rate of apoptotic cells after indicated        treatment)−(Rate of apoptosis of untreated cells)    -   V1, V2 and V3 were tested in 4 independent experiments. V4 and        V5 were tested in 2 independent experiments.

CONCLUSION

Our first results showed, that V1 and V5 were able to induce apoptosisin CD3/CD28 costimulated T Lymphocytes. V2, V3 and V4 were not able toinduce apoptosis. Comparing V1 and V5 mediated induction of apoptosiswith azathioprine or 6-mercaptopurine mediated induction of apoptosis,V1 and even V5 mediated effects seemed to be more pronounced andappeared earlier (FIGS. 2-4).

EXAMPLE 5 Preparation of 2′/3′-Methylenoaminocarbamate derivatives of6-Thio-Guanosine-triphosphate

The dry tributylammonium salt of 6-Thio-GTP 1 (0.2 mmol) was treatedwith 200 mg carbonyl-diimidazole in 4 ml dimethylformamide. Theresulting mixture was stirred for 6 hours at 0° C., brought to roomtemperature and, subsequently, 80 μl of methanol were added. After 10minutes, 2 mmol of the appropriate methyleno-amine and 2 ml oftriethylamine were also added to the reaction mixture. The solution wasstirred overnight at room temperature and the solvent was then removedunder reduced pressure. The residue was taken up in 30 ml water and themixture was adjusted to pH 1, in order to decompose the resultingintermediate phosphoramidate at the triphosphate moiety. After 20minutes, the solution was adjusted to pH 7.5, the precipitate filteredoff and the solvent was removed in vacuo. The resulting crude productwas purified by ion exchange chromatography and subsequently by reversedphase HPLC.

EXAMPLE 6 2′/3′-Methylenoaminocarbamate-6-Thio-Guanosine-triphosphatederivative 2a

Reaction of 3-thienyl-methylamine (226 mg, 2 mmol) with 1 according tothe general procedure yielded 2a (0.58 mmol, 29%) after purification byion exchange chromatography and subsequent reversed phase HPLC.

2′/3′-Methylenoaminocarbamate-6-Thio-Guanosine-triphosphate Derivative2b

Reaction of (1,5-Dimethyl-1H-pyrazol-3-yl)methylamine (250 mg, 2 mmol)with 1 according to the general procedure yielded 2b (0.66 mmol, 33%)after purification by ion exchange chromatography and subsequentreversed phase HPLC.

EXAMPLE 7 Preparation of 2′/3′-Carbamate Derivatives of6-Thio-Guanosine-triphosphate

The dry tributylammonium salt of compound 1 (0.2 mmol) was treated with200 mg carbonyl-diimidazole in 4 ml dimethylformamide. The resultingmixture was stirred for 6 hours at 0° C., brought to room temperatureand, subsequently, 80 μl methanol were added. After 10 minutes, 2 mmolof the appropriate amine and 2 ml of 1 M potassium hexamethyldisilazide(KHMDS) in THF were carefully added to the solution. The solution wasstirred at room temperature for 1 hour and the solvent was then removedunder reduced pressure. The residue was taken up in 30 ml water and themixture was adjusted to pH 1, in order to decompose the resultingintermediate phosphoramidate at the triphosphate moiety. After 20minutes, the solution was adjusted to pH 7.5, the precipitate filteredoff and the solvent was removed in vacuo. The resulting crude productwas purified by ion exchange chromatography and subsequently by reversedphase HPLC.

EXAMPLE 8

In relation to FIGS. 5 and 6, an overview of at least three experimentaldata sets on apoptosis is shown below. Here, some of the D compoundswere able to induce apoptosis. B0 was the strongest candidate drug forapoptosis induction. Two issues should be considered in these results.

-   -   Negative induction of apoptosis means that there were more        apoptotic cells in the untreated group than in the treated        group. This phenomenon may appear from time to time and may be        explained by a kind of statistical variance.    -   The drug 6-Thio-GTP on average was not able to induce apoptosis        in these experiments. Generally 6-Thio-GTP should be a positive        control to induce apoptosis in T cells. In these experiments the        positive control did not work very well. This might be explained        by the fact that the experiments are often preformed with        primary T cells, which are freshly isolated from blood of        different donors. It is well known, that some people are not        sensitive for azathioprine therapy. In this way, T cells of some        donors might be resistant against 6-Thio-GTP induced apoptosis.        In any case, however, B0 and some of the D compounds were able        to induce apoptosis suggesting that they are candidate drugs.

In addition, an alternative method was performed for screening ofgroup-D derivatives (FIG. 8). It was decided to analyse the activity ofcaspase-3/7 in T cells, which were treated with group-D derivatives. Ascompared to AnnexinV/PI staining this new method might have someadvantages:

-   -   It is an easier protocol. Therefore there are fewer        possibilities for individual errors.    -   Increased activity of caspase-3 is very specific for apoptosis.        Therefore this method is very sensitive for the detection of        apoptosis. There is no interfering influence of necrotic cells.    -   The measurement is done in duplicate. In this way there is an        internal control.

The Caspase-Glo 3/7 Assay (Promega™) was used. This assay is based on acaspase dependent luminescent signal. Protocol: CD4⁺ T cells wereisolated from human blood by magnetic beads (Dynal). T lymphocytes werestimulated in complete RPMI-1640 medium (RPMI-1640+10% FCS+100 U/mlPenicillin/Streptomycin+3 mM L-Glutamin) for 3 days with coatedantibodies to CD3 (0.04 μg/ml) and soluble CD28 antibodies (PharMingen™;1 μg/ml) plus IL-2 (R & D Systems™, Wiesbaden, Germany; 40 U/ml) in96-well plates. Cells were treated with different group-D derivatives orwere left untreated. At day three of culture Caspase-3/7 Assay wasperformed. 25 μl of Caspase-Glo 3/7 reagent was added to each well.Probes were mixed gently for 2 minutes and incubated at room temperaturefor 30 minutes. Finally 100 μl of each probe were transferred to a whitewalled 96-well luminometer plate and analysed in a plate-readingluminometer. The added reagent contains a specific substrate ofcaspase-3 and caspase-7. Cleavage of this substrate by activatedcaspase-3 results in luciferase reaction. Luminescence is proportionalto the amount of caspase activity present (FIG. 8)

EXAMPLE 8 2′/3′-Carbamate-6-Thio-Guanosine-triphosphate derivative 3a

Reaction of 3-aminopyrimidine (190 mg, 2 mmol) with 1 according to thegeneral procedure yielded 3a (0.076 mmol, 3.8%) after purification byion exchange chromatography and subsequent reversed phase HPLC.

EXAMPLE 9 2′/3′-Carbamate-6-Thio-Guanosine-triphosphate derivative 3b

Reaction of 3-(tert.-Butyl)-1-methyl-1H-pyrazol-5-amine (306 mg, 2 mmol)with 1 according to the general procedure yielded 3b (0.11 mmol, 5.5%)after purification by ion exchange chromatography and subsequentreversed phase HPLC.

BIBLIOGRAPHY

-   -   Boise L H et al. Receptors that regulate T-cell susceptibility        to apoptotic cell death. Ann N Y Acad Sci 1995; 766:70-80.    -   Tiede I. et al. CD28-dependent Rac1 activation is the molecular        target of azathioprine in primary human CD4+ T lymphocytes. J        Clin Invest 2003; 111: 1133-1145.    -   Maltzman J S et al. Azathioprine: old drug, new action. J Clin        Invest 2003; 111: 1122-1124.    -   Boise L H et al. CD28 costimulation can promote T cell survival        by enhancing the expression of bcl-x_(L). Immunity 1995; 3:        87-98.    -   Khoshnan A. et al. The NF-κB cascade is important in bcl-x_(L)        expression and for the anti-apoptotic effects of CD28 receptor        in primary human CD4+ T lymphocytes. J Immunol 2000; 165:        1743-1754.    -   Noel P J et al. CD28 costimulation prevents cell death during        primary T cell activation. J Immunol 1996; 157: 636-642.    -   Frauwirth K A et al. Activation and inhibition of lymphocytes by        costimulation. J Clin Invest 2002; 109: 295-299.    -   Marinari B et al. Vav cooperates with CD28 to induce NF-κB        activation via a pathway involving Rac1 an mitogen-activated        kinase 1. Eur J Immunol 2002; 32: 447-456.    -   Faruqi T R et al. Rac1 mediates STAT-3 activation by autocrine        IL-6. PNAS 2001; 98: 9014-9019.    -   Mudter J and Neurath M F. The role of signal transducers and        activators of transcription in T inflammatory bowel diseases.        IBD 2003; 9: 332-337.    -   Lovato P et al. Constitutive STAT-3 activation in intestinal T        cells from patients with Crohn's disease. J Biol Chem 2003; 278:        16777-16781.    -   Van Aelst L et al. Rho GTPases and signaling networks. Genes &        Development 1997; 11: 2295-2322.    -   Kohyoma et al. (2003) A facile synthesis of AICAR from inosine.        Synthesis 17:2639.    -   Imai et al. (1971) Synthesis of compounds related to inosine        5-phosphate and their flavor enhancing activity. IV        2-substituted inosine %′-phosphates. Chem. Pharm. Bull. 19:576.    -   Ostermann et al (1999), New N-2-labelled fluorescent derivates        of guanosine nucleotides and their interaction with GTP-binding        proteins. Nucleosides & Nucleotides 18:245.    -   Ludwig (1981) Acta Biochim. Acad. Sci. Hung. 16:131

1. A compound of the general formula (I):

wherein the dashed bond in the sugar moiety can be either single ordouble and wherein R1, R2, R3, R4 or R5, equal or different between eachother, have general formula -(Int)_(m)-Ter, wherein m is between 0 and12 and Int and Ter are Internal and Terminal building blocks, whereinInt is selected from the group consisting of

and Ter is selected from the group consisting of

and wherein X represents either carbon or nitrogen atom within aromaticring, Y represents either oxygen or sulphur atom and an additional groupQ, group Qi or groups Qi (Qi indicates that the group or several groupsmay be bound to any unsaturated moiety of the ring) are selected fromthe group consisting of —OH, —COOH, —N(CH₃)₂, —N(CH₂—CH₃)₂, —CO—CH₃,—CO—O—CH₃, —O—CH₃, —S—CH₃, —SO₂—CH₃, —CN, —NO₂ or -halogen elements andwherein R5 may be

and metal and ammonium salts thereof, wherein n is between 0 and 5, oroxygen or phosphorus is partially or completely replaced by nitrogen,sulphur, methyleno groups or their derivatives.
 2. A compound accordingto claim 1 wherein Int is selected from the group consisting of


3. A compound according to claim 1 wherein Ter is selected from thegroup consisting of


4. A compound according to claim 1 wherein said compounds are labelled.5. A compound according to claim 1 with the general formula (Ia):

wherein R₁, R₂, R₃, R₄ or R₅, equal or different between each other,have general formula -(Int)_(m)-Ter, wherein m is between 0 and 12 andInt and Ter are Internal and Terminal building blocks, wherein Int isselected from the consisting of

and Ter is selected from the consisting of

wherein an additional group Q, group Qi or groups Qi (Qi indicates thatthe group or several groups may be bound to any unsaturated moiety ofthe ring) are selected from the group consisting of —OH, —COOH,—N(CH₃)₂, —N(CH₂—CH₃)₂ or -halogen elements
 6. A compound according toclaim 1, wherein sugar moiety of compounds of formula (I) are selectedfrom the group consisting of the following sugar moieties or sugar-likemoieties:


7. A compound according to claim 1 wherein R₃ or R₄ are selected from

and wherein Q is selected from —OH (FAM) or —N(CH₃)₂ (TAMRA).
 8. Acompound as claimed in claim 1 with the following structure:


9. A compound as claimed in claim 1 with the following structure:


10. A compound as claimed in claim 1 with the following structure:


11. A compound as claimed in claim 1 with the following structure:


12. A compound as claimed in claim 1 with the following structure:


13. A compound as claimed in claim 1 with the following structure:


14. A compound as claimed in claim 1 with the following structure:


15. A compound as claimed in claim 1 with the following structure:


16. A compound as claimed in claim 1 with the following structure:


17. A compound as claimed in claim 1 with the following structure:


18. A compound as claimed in claim 1 with the following structure:


19. A compound according to claim 1 of the general formula (II):

wherein n=1, 2 or 3, m is between 0 and 5, Int is selected from thegroup consisting of

and Ter is selected from group the consisting of

wherein X represents either carbon or nitrogen atom within aromaticring, Y represents either oxygen or sulphur atom and an additional groupQ or groups Qi (i indicating the position of any unsaturated moiety ofthe ring to which the group Q may be bound) are selected from the groupconsisting of —CH₃, —C(CH₃)₃, —OH, —COOH, —CO—CH₃, —CO—O—CH₃, —O—CH₃,—S—CH₃, —SO₂—CH₃, —N(CH₃)₂, —N(CH₂—CH₃)₂, —CN, —NO₂ or -halogenelements.
 20. A compound according to claim 19 wherein, Ter is selectedfrom the group consisting of


21. A compound as claimed in claim 19 wherein n is 1-3.
 22. A compoundas claimed in claim 19 wherein n is
 3. 23. A compound as claimed inclaim 19 wherein n is
 1. 24. A compound as claimed in claim 19 wherein nis
 2. 25. A compound as claimed in claim 19 with the following generalformula:


26. A compound as claimed in claim 19 with the following generalformula:


27. A compound as claimed in claim 19 with the following generalformula:


28. A compound as claimed in claim 19 with the following generalformula:


29. A compound as claimed in claim 19 with the following generalformula:


30. A compound as claimed in claim 19 with the following generalformula:


31. A compound as claimed in claim 19 with the following generalformula:


32. A compound as claimed in claim 19 with the following generalformula:


33. A compound according to claim 1, wherein said compounds are selectedfrom the group consisting of 2′,3′-EDA-6-Thio-GTP,FAM-2′,3′-EDA-6-Thio-GTP, TAMRA-2′,3′-EDA-6-Thio-GTP,Aspartate-2′,3′-EDA-6-Thio-GTP, Glutamate-2′,3′-EDA-6-Thio-GTP,Threonine-2′,3′-EDA-6-Thio-GTP, Serine-2′,3′-EDA-6-Thio-GTP,2′,3′,5′,O-Triacetyl-N-2-(Acetyl-6″-aminohexyl)-guanosine,2′,3′,5′-Triacetyl-N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine,N-2-(6″-thioacetamide-hexyl)-6-Thioguanosine,N-2-(6″-Aminohexyl)-6-Thioguanosine,N-2-(6″-guanidino-hexyl)-6-Thioguanosine,N-2-(6″-Aminohexyl)-6-Thio-GMP, N-2-(6″-guanidino-hexyl)-6-Thio-GMP,N-2-(6″-Aminohexyl)-6-Thio-GTP, N-2-(6″-guanidino-hexyl)-6-Thio-GTP,N-2-(6″-Aspartate-hexyl)-6-Thioguanosine,N-2-(6″-Glutamate-hexyl)-6-Thioguanosine,N-2-(6″-Threonine-hexyl)-6-Thioguanosine,N-2-(6″-Serine-hexyl)-6-Thioguanosine, N-2-(6″-Aminobutyl)-6-Thio-GTP,N-2-(6″-guanidino-butyl)-6-Thioguanosine,N-2-(6″-Aspartate-butyl)-6-Thioguanosine,N-2-(6″-Glutamate-butyl)-6-Thioguanosine,N-2-(6″-Threonine-butyl)-6-Thioguanosine,N-2-(6″-Serine-butyl)-6-Thioguanosine,N-2-(6″-Aminopropyl)-6-Thioguanosine,N-2-(6″-guanidino-propyl)-6-Thioguanosine,N-2-(6″-Aspartate-propyl)-6-Thioguanosine,N-2-(6″-Glutamate-propyl)-6-Thioguanosine,N-2-(6″-Threonine-propyl)-6-Thioguanosine,N-2-(6″-Serine-propyl)-6-Thioguanosine,N-2-(6″-Amino-2-butene)-6-Thioguanosine,N-2-(6″-guanidino-2-butene)-6-Thioguanosine,N-2-(6″-Aspartate-2-butene)-6-Thioguanosine,N-2-(6″-Glutamate-2-butene)-6-Thioguanosine,N-2-(6″-Threonine-2-butene)-6-Thioguanosine,N-2-(6″-Serine-2-butene)-6-Thioguanosine,N-2-(6″-Amino-2-butyne)-6-Thioguanosine,N-2-(6″-guanidino-2-butyne)-6-Thioguanosine,N-2-(6″-Aspartate-2-butyne)-6-Thioguanosine,N-2-(6″-Glutamate-2-butyne)-6-Thioguanosine,N-2-(6″-Threonine-2-butyne)-6-Thioguanosine,N-2-(6″-Serine-2-butyne)-6-Thioguanosine,N-2-(6″-Amino-2,4-hexadiyne)-6-Thioguanosine,N-2-(6″-guanidino-2,4-hexadiyne)-6-Thioguanosine,N-2-(6″-Aspartate-2,4-hexadiyne)-6-Thioguanosine,N-2-(6″-Glutamate-2,4-hexadiyne)-6-Thioguanosine,N-2-(6″-Threonine-2,4-hexadiyne)-6-Thioguanosine,N-2-(6″-Serine-2,4-hexadiyne)-6-Thioguanosine.
 34. A pharmaceuticalcomposition comprising at least one of the compounds as claimed in claim1 as active principle and one or more pharmaceutically acceptableco-adjuvants or excipients.
 35. (canceled)
 36. A method of preventingrejection of organ transplants or of post-transplant nephropathy in anindividual in need thereof comprising administering to the individual acompound of claim
 1. 37. The method of claim 36 further comprisingadministering radiotherapy, corticosteroids or cytotoxic agents to theindividual.
 38. A method of treating one or more pathologies in anindividual in need thereof, wherein the pathologies are selected fromthe group consisting of inflammatory chronic intestinal diseases,auto-immune enteropathy, active chronic hepatitis, rheumatoid arthritis,Still's disease, systemic lupus erythematous, acquired haemolyticanaemia, idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis,polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoidnephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris,primary biliary cirrhosis, primary sclerosing cholangitis, recurrentmultiform erythema, chronic actinic dermatitis, gangrenous hypoderm,ptyriasis rubra, Wegener's granulomatosis, cutaneous vasculitis, atopicdermatitis, psoriasis, pimply pemphigoid, comprising administering tothe individual a compound of claim
 1. 39. The method of claim 38,wherein inflammatory chronic intestinal diseases are selected from thegroup consisting of Crohn's disease, ulcerous rectocolitis,indeterminate colitis.
 40. The method of claim 38 further comprisingadministering radiotherapy, corticosteroids or cytotoxic agents to theindividual.
 41. A method for the treatment of cancer in an individual inneed thereof, comprising administering to the individual a compound ofclaim
 1. 42. A method of evaluating the binding properties of thecompounds of formula (I) by the RacI/Vav system wherein a compound ofclaim 1 is used in the RacI/Vav system.
 43. (canceled)
 44. The method ofclaim 36 wherein the organ transplants include kidney, heart, lung,pancreas and liver transplantation.
 45. A process for the preparation ofa compound as claimed in claim 1 wherein the introduction of the —NH—Rgroup at the 2 position of guanosine ring comprises the following steps:a) protection of the NH moiety of tri-O-acetyl-inosine; b) oxidativeguanosine ring-opening and O-deprotection; c) guanosine ring-closing andintroduction of a SH group at the 2 position of the guanosine ringthrough the use of CS₂; d) replacing the SH group at the 2 position withan amino-linker by using an excess of an aliphatic diamine.
 46. Processaccording to claim 45, comprising a further step e) of protection ofribose OH groups and of the primary amine group by acetylation. 47.Process according to claim 46, comprising a further step f) ofthiolation of C═O groups through the use of Lawesson's reagent. 48-52.(canceled)